Many heterologous proteins are not capable of being expressed in Escherichia coli in any measurable yield, or even if detectable, are not capable of being expressed at such commercially recoverable levels due to instability of the foreign protein in the host. Small proteins (e.g., peptide hormones of less than 100 amino acids) appear to be especially sensitive to degradation or host proteolysis. The degree of instability varies from host to host and protein to protein.
Thus, in many situations, for reasons which have not been completely resolved, heterologous proteins, despite the use of active transcriptional promoters and high copy number plasmids, are produced in only minor amount, or not at all, in a microorganism host. Moreover, as described in greater detail herein, expression systems which are quite adequate for laboratory scale expression of a desired protein are not suitable for high-density fermentation.
One of the ways in which instability or proteolytic degradation problems have been resolved employs the use of a DNA sequence encoding an additional protein which, on its own, is stable in the host cell and joining this sequence to the coding sequence of the desired protein. A small number of prokaryotic proteins have been used as the fusion partner in this manner: E. coli lacZ, trpE, chloramphenicol acetyl transferase (CAT), recA and lambda cII, for example.
While the literature establishes that fusion proteins are useful to express heterologous proteins in bacteria, efforts to use mammalian proteins as the fusion partner to express or to increase the recoverable yield of heterologous proteins have not been reported. More particularly, there is no reported use of human connective tissue-activating peptide-III (also referred to herein as CTAP-III) as one component in a fused protein construct to stabilize the expression of a poorly expressed or unstable protein. In light of the fact that many important proteins cannot be successfully expressed in bacteria in any commercially recoverable yield, there is a need to develop systems suitable for high density fermentation for the bacterial expression and recovery of such proteins.